Based on expression analysis of mouse type A spermatogonia, pachytene spermatocytes, and round spermatids we cloned a novel mArd1 (Arrest Defective 1) homolog, which we named mArd2 (also known as Ard1b) that demonstrated testis-specificity and elevated expression in pachytene spermatocytes. The mArd1 protein is known to interact with an auxiliary protein subunit mNAT1 to constitute a functional N-acetyltransferase. We showed that the protein encoded by mArd2 is functionally similar to its homolog mArd1 in vitro. However, the two homologous genes displayed a grossly different expression patterns in various cell lines and mouse tissues. Specifically, the mArd2 gene is expressed in a developmentally regulated manner during spermatogenesis. To elucidate the molecular mechanism of the tissue-specificity of mArd2, an mArd2 promoter assay was performed in two mArd2 non-expressing cell lines, namely NIH/3T3 and GC-2spd(ts), which represent cells of somatic and early germ-line origin, respectively. The detection of mArd2 promoter activities in both cell types indicates that the transcriptional machinery required for mArd2 expression is intact; the absence of mArd2 expression would thus be the result of other regulatory mechanisms. We hypothesize that DNA methylation could play a role in the regulation of mArd2 expression. [unreadable] [unreadable] With 5-aza-deoxycytidine treatment in non-expressing cells, in vitro methylation and bisulfite sequencing analysis, we showed that mArd2 transcription is regulated epigenetically. Comparison of the methylation status of the two CpG islands spanning the proximal promoter and half of the coding region of mArd2 gene showed that the proximal promoter of mArd2 was hypermethylated in mouse tissues that do not express mArd2 but hypomethylated in male germ cells which express the gene. On the other hand, the distal CpG island was found to be hypomethylated in germ cells at stages that display higher mArd2 expression, but hypermethylated in non-expressing tissues as well as mitotic germ cells in which mArd2 expression is minimal. Thus, our findings confirmed the hypothesis that the testis-specific expression of mArd2 is epigenetically regulated. The Ard1-Ard2 expression system could be a paradigm for studying epigenetic regulation of mammalian spermatogenic gene expression. [unreadable] [unreadable] In spite of these findings, the involvement of other protein factors in the activation of Ard2 transcription in testis remains unknown. We attempt to address this question by identifying the minimal Ard2 promoter. Our data indicated that the minimal promoter of Ard2 is proximal to the transcriptional start site of the gene and overlapped with one of the CpG islands in the gene. We are in the process of isolating the factors that are bound to this sequence so as to understand how activation and suppression of Ard2 transcription is regulated. [unreadable] [unreadable] We also demonstrated the translation of Ard2 in vivo was delayed during spermatogenesis, with the transcript level peaked in meiotic germ cells but the protein was not expressed until after round spermatids appeared. We hypothesized the 3 untranslated region (UTR) of Ard2 conferred a repressive effect on its translation. We established a cell culture model to test the ability of the UTR to repress reporter gene expression. From this we identified several regions on the 3 UTR that are involved in the suppression of reporter translation. We are isolating the testicular protein factors that would be bound to these sequences. The involvement of such protein factors in regulating the translation of other spermatogenic transcripts would also be studied. [unreadable] [unreadable] Finally, the global effect of a deficiency of protein N-alpha terminal acetylation on cell functions is also being studied by gene-knockdown experiments in mammalian cells. [unreadable] [unreadable] A second gene that we have studied in the past year is Lin28. Lin28 is a heterochronic gene involved in the temporal control of cell fate determination in C. elegans. In mammals, Lin28 is mostly detected in cells that possess proliferative capacity, e.g. embryonic stem cells, embryonal carcinoma cells and mouse type A spermatogonia. Lin28 is known to be localized predominantly in cytoplasm. However, a recent study showed that Lin28 is involved in microRNA maturation process in the nucleus. These observations suggest that Lin28 may have dual role in the cell by interacting preferentially with nuclear and cytoplasmic proteins, respectively. However, its biological function, especially its role in mammalian development, is largely unknown. [unreadable] [unreadable] We hypothesized Lin28 would act as an RNA chaperone to control the availability of transcripts that encode functions which are important to cell fate determination. In a cell culture model, we have identified a specific set of RNA transcripts that are bound by Lin28. The corresponding change in protein levels of these transcripts is being studied by gene knockdown experiment. On the other hand, we also isolate the proteins that interact with Lin28 in the nuclear and cytoplasmic fractions of expressing cells in order to examine the biochemical role of Lin28. [unreadable] [unreadable] The selectivity of gene expression in certain cell types suggests that the transcription of Lin28 is strictly regulated. Our preliminary data showed that Lin28 transcription is not regulated by DNA methylation. Instead, a specific genomic region about one kilo bp upstream of the transcriptional start site showed promoter activity only in Lin28-expressing cells. We are screening a panel of Lin28 expressing and non-expressing cells to identify the genetic factors that are involved in activating or suppressing Lin28 transcription. [unreadable] [unreadable] Lastly, through algorithmic analysis of mouse tiling array signals of the 3 main cell stages of spermatogenesis with reference sequences, a pool of stage specific genes were identified. Transmembrane and coiled-coil domain 5A (Tmco5A) and 4930563P21Rik (Tmco5B) were two of the novel genes identified which express exclusively in mouse testis during the post-meiotic stage of male germ cell development. Both genes have a single CAGE tag in testis. Comparative genomics showed conservation of the 2 genes in different species, including human. Evidence from protein structure prediction, gene mapping, sequence alignment and expression profiles suggested that Tmco5A and Tmco5B are very likely to belongto a single gene family with a specific role in male post-meiotic development or sperm function. Our studies are aimed at characterization of the two genes in mouse testis.